A missense variant at the RAC1-PAK1 binding site of RAC1 inactivates downstream signaling in VACTERL association

RAC1 at 7p22.1 encodes a RAC family small GTPase that regulates actin cytoskeleton organization and intracellular signaling pathways. Pathogenic RAC1 variants result in developmental delay and multiple anomalies. Here, exome sequencing identified a rare de novo RAC1 variant [NM_018890.4:c.118T > C p.(Tyr40His)] in a male patient. Fetal ultrasonography indicated the patient to have multiple anomalies, including persistent left superior vena cava, total anomalous pulmonary venous return, esophageal atresia, scoliosis, and right-hand polydactyly. After birth, craniofacial dysmorphism and esophagobronchial fistula were confirmed and VACTERL association was suspected. One day after birth, the patient died of respiratory failure caused by tracheal aplasia type III. The molecular mechanisms of pathogenic RAC1 variants remain largely unclear; therefore, we biochemically examined the pathophysiological significance of RAC1-p.Tyr40His by focusing on the best characterized downstream effector of RAC1, PAK1, which activates Hedgehog signaling. RAC1-p.Tyr40His interacted minimally with PAK1, and did not enable PAK1 activation. Variants in the RAC1 Switch II region consistently activate downstream signals, whereas the p.Tyr40His variant at the RAC1-PAK1 binding site and adjacent to the Switch I region may deactivate the signals. It is important to accumulate data from individuals with different RAC1 variants to gain a full understanding of their varied clinical presentations.


Results
Clinical features. Detailed clinical features of our patient are shown in Table 1 and Fig. 1. The proband (II-3) was born from his mother's third pregnancy. Fetal ultrasonography at 28 weeks of gestation identified multiple congenital anomalies: total anomalous pulmonary venous return (Fig. 1a), enlarged coronary sinus (Fig. 1b) and a fourth vessel in the three vessel and trachea view (Fig. 1c) implying persistent left superior vena cava, scoliosis (Fig. 1d), and right-hand polydactyly. A small stomach bubble ( Fig. 1e) with polyhydramnios implied esophageal atresia. The fetal karyotype by amniocentesis was normal (46,XY). An emergency cesarean section was performed at 30 weeks and 3 days of gestation because of non-reassuring fetal status. His birth weight was 1406 g (− 0.69 SD) and Apgar scores were 3 (pulse: 2, activity: 1, others: 0) at 1 and 5 min after birth, respectively.
Multiple anomalies were confirmed at birth, including scoliosis (Fig. 1f), tracheal agenesis type III (Fig. 1g), low set ears, micrognathia, and right-hand polydactyly (Fig. 1h). He died of respiratory failure caused by tracheal agenesis type III (Fig. 1i,j) on the first day after delivery. Prenatally suspected esophageal atresia was determined to be esophagotracheal fistula at autopsy (Fig. 1i). His autopsy additionally revealed that he had 10th thoracic vertebra hypoplasia, 6th and 7th rib fusion, and symmetrical liver ( Supplementary Fig. S1a). Other autopsy findings are presented in Supplementary Fig. 1. These clinical features were compatible with VACTERL association 15 .

Identification of a pathogenic RAC1 variant. Exome sequencing identified a missense variant in RAC1
[NM_018890.4:c.118T > C p.(Tyr40His)] in the proband (II-3). Trio-based Sanger sequencing confirmed that this variant occurred de novo (it was not found in the biological parents, which was confirmed with microsatellite markers) (Fig. 2a) and was absent from our in-house exome database of 575 Japanese individuals and other control population databases. This variant is in exon 3 of RAC1, which encodes the RAC1-PAK1 binding site 16 and part (4 amino acids) of the Switch I region (Fig. 2b). Many other pathogenic variants are clustered in exon 3 and Tyr40 is highly evolutionarily conserved among vertebrates (Fig. 2b). Consistently, this variant was judged as disease-causing by SIFT, PolyPhen-2, MutationTaster, and CADD (Table 1). This variant was also classified as a likely pathogenic variant based on the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines [PS2 (de novo), PM1 (located in hot spot), PM2 (absent from control), and PP3 (computational evidence)] 17 .
Biochemical properties of RAC1-p.Tyr40His. To investigate the properties of RAC1-p.Tyr40His, we examined its activation status. GTP/GDP-exchange and GTP-hydrolysis activities of this variant were compared with those of wild-type RAC1. When log 10 k obs (observed rate constant) was measured, the exchange activity of RAC1-p.Tyr40His was comparable to that of wild-type RAC1 (Fig. 3a,b). Notably, the exchange reaction of the variant with Trio-D1 (a GEF) was promoted to a level similar to that of the wild type (Fig. 3a,b). However, GTP-hydrolysis activity of RAC1-p.Tyr40His was significantly decreased compared with that of wild-type RAC1 (Fig. 3c,d). Meanwhile, α-Chimerin (a GAP) efficiently increased the hydrolysis activity, similarly to the wild type (Fig. 3c,d). Overall, therefore, RAC1-p.Tyr40His demonstrated a slightly lower GTP-hydrolysis activity than wild-type RAC1, while GTP/GDP-exchange activity was similar to that of the wild type. We assume that while RAC1-p.Tyr40His on its own may be a moderately activated version in vitro, it interacts with GEFs and GAPs and cycles between GDP-bound inactive and GTP-bound active states.
Interaction of RAC1-p.Tyr40His with PAK1, a downstream effector. Effects of the RAC1-p.
Tyr40His variant on interaction with downstream signaling pathways were examined. We focused on PAK1, one of the most characterized RAC1 effector molecules, which is involved in actin cytoskeletal reorganization, cell adhesion and cell signaling 18 . We performed pull-down assays to evaluate the interaction of RAC1-p. Tyr40His with the PAK p21-binding domain (PAK1-PBD) 19 . RAC1-p.Tyr40His bound minimally with PAK1-PBD (Fig. 4a,b and Supplementary Fig. S2a-c). Given the biochemical analysis results (Fig. 3a- www.nature.com/scientificreports/ Tyr40His on its own was presumed to be in a preferentially inactive GDP-bound state in COS7 cells (monkey kidney fibroblast-like cells). We then asked whether RAC1-p.Tyr40His interacts with PAK1 under the GTPbound status. It is notable that RAC1-p.Tyr40His activated by Trio-D1 did not interact with PAK1-PBD under the conditions in which activated wild-type RAC1 efficiently interacted with PAK1-PBD (Fig. 4a,b and Supplementary Fig. S2a-c). Considering that GTP loading on the variant occurred normally in vitro (Fig. 3a,b), RAC1-p.Tyr40His was predicted to undergo GTP-dependent conformational change in response to Trio-D1 but it failed to form a complex with PAK1-PBD. We concluded that RAC1-p.Tyr40His did not activate PAK1 even in the biochemically "active" GTP-bound form.
We then tried to confirm the effects of the p.Tyr40His variant on PAK1 activity. To this end, RAC1-p.Tyr40His was expressed with Myc-PAK1 and Trio-D1 in COS7 cells, and PAK1 activation status was analyzed based on its autophosphorylation at Ser199 and Ser204 20 . Consequently, PAK1 was found to be minimally activated by RAC1-p.Tyr40His with Trio-D1, under the conditions where wild-type RAC1 activation caused PAK1 autophosphorylation (Fig. 4c,d, and Supplementary Fig. S2d-i). Trio-D1-mediated PAK1 activation without RAC1 may have occurred because of endogenous RAC proteins (Fig. 4c,d, and Supplementary Fig. S2g-i). These results Table 1. Clinical features of the current patient and a previously reported patient with RAC1 variants adjacent to the Switch I region. NA not available, PLSVC persistent left superior vena cava, SD standard deviation, TAPVR: total anomalous pulmonary venous return. The pathogenicity of each prediction score was judged according to the following values: SIFT < 0.05 (Deleterious), PolyPhen-2 0.65-1.0 (Damaging), and CADD (phred score) ≥ 20 (Pathogenic). a Reijnders et al. 13

Structural considerations for RAC1 variants adjacent to the Switch I region.
Based on the crystal structure of human RAC3 (which is highly homologous to RAC1) complexed with an effector, PAK1 (PDB: 2QME), we evaluated the structural effects of the p.Tyr40His variant and compared them with the previously reported human RAC1 p.Asn39Ser variant 13 . Asn39, Tyr40, and their interacting residues in RAC1 are fully conserved in RAC3.
In this complex, Tyr40 of RAC3 is in the binding region for PAK1 and adjacent to the Switch I region (Thr25-Asn39) (Fig. 5a, left and middle). Tyr40 makes a hydrogen bond with Asp57. It also forms a hydrophobic core with Leu20, Ile21, and Asp38 (π orbitals) of RAC3, and Phe17 and His19 of PAK1, playing an important role in forming the RAC3-PAK1 complex (Fig. 5a, middle). Consequently, the p.Tyr40His variant would break the hydrogen bond and destabilize the hydrophobic core (Fig. 5a, right), resulting in impaired complex formation.
Asn39 also contributes to RAC3-PAK1 complex formation by making a network of hydrogen bonds with Thr20 of PAK1 and the main chain oxygen of Leu55 of RAC3 and also van der Waals contacts with Trp56 of RAC3 (Fig. 5b, left and middle). Based on the p.Asn39Ser variant model, Ser39 may maintain the hydrogen bond www.nature.com/scientificreports/ with Thr20 of PAK1 but could not interact with the main chain of Leu55 and Trp56 of RAC3 (Fig. 5b, right). This variant would therefore destabilize complex formation. The value of the free energy change for each variation calculated by FoldX 21,22 was significant for p.Tyr40His (3.75 ± 0.21 kcal/mol) and mild for p.Asn39Ser (0.95 ± 0.01 kcal/mol) (Fig. 5c), supporting our structural predictions.

Discussion
In this study, a de novo RAC1 variant [NM_018890.4:c.118T > C p.(Tyr40His)] located at the RAC1-PAK1 binding site, which is adjacent to the Switch I region, was identified in a Japanese patient. Compared with previously reported cases, this patient presented with very severe clinical features compatible with VACTERL association 15 , including cardiovascular anomalies, tracheoesophageal malformation, and skeletal anomalies, and died of respiratory failure on the first day after delivery at 30 weeks and 3 days of gestation.
All previously reported cases with RAC1 variants located in Switch I or II regions involved altered GTP/ GDP-bound states 13,14,23 . In the Online Mendelian Inheritance in Man (https:// omim. org/), de novo RAC1 variants have been found in intellectual developmental disorder (MIM#617751) with the highly variable phenotype, such as neurodevelopmental delay with abnormal brain magnetic resonance imaging findings, epilepsy, facial dysmorphisms, cardiovascular malformations and poor feeding. However, our case was more clinically severe with lethal malformations. Therefore, considering possible other variants, we checked the patient's exome data but found no other deleterious variants (including single nucleotide variants and copy number variants).
To obtain insight into the pathophysiological mechanism of the p.Tyr40His variant, we performed in vitro characterization. Biochemical analyses revealed that this variant exhibited GTP/GDP-exchange activity comparable with that of wild-type RAC1 and GTP-hydrolysis activity only slightly affected. Therefore, the effect of this variant on biochemical properties did not seem to be significant. Given that the p.Tyr40His variant occurs at a residue adjacent to the Switch I region but not in the G-domain, which is essential for GTP/GDP-exchange and GTP-hydrolysis activities (Fig. 2b), it is plausible that this variant only slightly affected the biochemical properties of RAC1 (Fig. 3a-d). From these results, we consider that RAC1-p.Tyr40His preferentially binds GDP in the resting state and converts to the GTP-bound form in response to upstream signaling.
We next focused on the interaction of the p.Tyr40His variant with effector molecules. The etiology of VACTERL association is unclear, but abnormalities in Hh, fibroblast growth factor and NOTCH signaling are thought to be involved in its pathogenesis 24,25 . Recently, Tang et al. showed RAC1-PAK1 pathway-mediated Hh signaling via translocation of Gli into the nucleus followed by its transactivation using mouse embryonic fibroblasts 9 . We therefore selected PAK1 as a representative effector for RAC1. Notably, despite its apparently normal biochemical properties, RAC1-p.Tyr40His was neither able to interact with nor activate PAK1 even www.nature.com/scientificreports/ in the presence of a GEF (Fig. 3a-d, and Supplementary Fig. S3), when the variant is supposed to be an active GTP-bound form. We assume that this is because of the position of the p.Tyr40His variant; it is located in the effector-interaction region (amino acids 32-41) of RAC1, which overlaps with the Switch I region (amino acids 25-39) (Fig. 2b) and, therefore, affects affinity toward downstream effectors. Given that there are ~ 30 RAC1 effectors, the p.Tyr40His variant should show a specific spectrum of interactions with effectors, as is the case for RAC3 26,27 , leading to variant-specific clinical and pathological phenotypes. Other as yet unanalyzed downstream effector system(s) may also be hampered by the p.Tyr40His variation; however, it is possible that RAC1-p. Tyr40His normally activates other downstream effector(s). It should be noted that the GTP-bound form of a small GTPase is generally recognized to be not only biochemically but also biologically activated and to activate downstream effectors. However, the results in Fig. 4 show that the GTP-bound state of the p.Tyr40His variant is not necessarily biologically active. We assume that RAC1p.Tyr40His acts as a dominant-negative version for PAK1-mediated signaling, suggesting that the RAC-PAK1 signaling axis may partially underlie the pathophysiology of the p.Tyr40His variant (Supplementary Fig. S3). In this context, a variant of the neighboring residue, p.Asn39Ser, was also reported to be a dominant-negative version based on the lamellipodia formation assay using fibroblastic cells 13 . It should, however, be noted that, in addition to the PAK1 signaling, this variant is likely to disrupt multiple downstream pathways because a range of other RAC1 effectors are known to bind via their common CRIB domains with RAC1 ( Supplementary Fig. S3) 28 . Further analyses are needed to uncover the precise pathophysiological mechanism of the p.Tyr40His variant.
The structural modeling studies also support our hypothesis that the p.Tyr40His variant inactivates RAC3-PAK1 signaling. Previous functional analyses of variants in the Switch II region showed constitutive activation of downstream signals in the RAC1-PAK1 axis 14 , and that variants adjacent to and in the Switch I region (p.Tyr40His and p.Asn39Ser) inactivate the signal. p.Tyr40His appeared to affect RAC3-PAK1 binding more strongly than p.Asn39Ser from the molecular modelling (Fig. 5c); therefore, it is reasonable that our p.Tyr40His case showed more severe phenotypes than the p.Asn39Ser case (Table 1).
In summary, we identified a novel RAC1 variant in a patient with a severe and lethal phenotype. We showed that the variant, which is located at the RAC1-PAK1 binding site, may inactivate the downstream pathway. Further analyses of additional patients are needed to determine the effects of other pathogenic variants on other downstream genes that contribute to VACTERL association. p-value was calculated as in (Fig. 3b). ***p < 0.001; **p < 0.01; *p < 0.05; n.s. not significant.

Methods
Subjects. The proband was born from the third pregnancy of healthy Japanese non-consanguineous parents.
The previous two pregnancies resulted in miscarriages, each having chromosomal abnormalities: trisomy 21 in the first and trisomy 19 in the second. Written informed consent was obtained from the patient's parents, in accordance with Japanese regulatory requirements. This study was approved by the Institutional Review Boards of Yokohama City University Faculty of Medicine under number A170525011 (modified B211100023) and Juntendo University Graduate School of Medicine (approval number 2017035). All experiments were performed in accordance with relevant guidelines and regulations. This practice was performed in accordance with the Declaration of Helsinki.
Structural considerations for the RAC1 variant. We referred to the crystal structure of human RAC3, which is highly homologous to RAC1, complexed with an effector, PAK1 (PDB: 2QME), to evaluate and compare the structural effects of the novel missense variant found in this study, RAC1-p.Tyr40His, with those of the known variant, p.Asn39Ser 13 . We used the program FoldX 21

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. All computational tools/codes and datasets used in this study can be downloaded through the following websites. ANNOVAR: http:// annov ar. openb ioinf ormat ics. org/ en/ latest/